Fig 2: Validation and effects of STAT5 knock down on miR-21 expression in K562 cellsCells were transduced at a MOI of 5 with either STAT5 shRNA or control shRNA lentiviral vector and GFP+ cells were sorted 48h later. (A) The expression of STAT5 was assessed by western blot analysis, confirming the efficacy of the STAT5 shRNA. (B, C) MiR-21 was quantified in the transduced cells either directly by RT-qPCR (B) or indirectly (C) using a reporter “sensor” plasmid driving the transcription of luciferase cds followed by two miR-21 binding sites. (D) The knock-down of STAT5 decreased the miR-21 promoter activity in cells transfected with the miR-21 promoter-luciferase plasmid and abolished the imatinib-induced effect. ***P<0.001, ** P <0.01 (control shRNA versus STAT5 shRNA); # P <0.05 (imatinib-treated versus not treated cells).

Fig 3: Effect of TKI treatment on the expression of miR-21 and PDCD4 in AML cells lines(A) MV4.11 and MOLM13 cells were treated with sunitinib (0.1µM, 24h). Then, PDCD4 mRNA and miR-21 were quantified by RT-qPCR (A), and miR-21 promoter activity was assessed by luciferase measurements in cells transduced with the miR-21 promoter-Luciferase lentivirus. (B) For each cell line, histograms represent the base 2 logarithm of the ratio (treated/not treated cells). (C) phospho-STAT5 levels were assessed in control (-) or TKI-treated (+) MV4.11 (left) and MOLM13 (right) cells. (D) Luciferase activity was measured in MOLM13 cells 24 hours after transfection with pGL4.10 plasmids having no promoter, WT or mutated miR-21 promoters.

Fig 4: NPM1 physically associates with STAT5. (a) Lysates from TF-1 cells (4 × 106 cells per condition) maintained in GM-CSF were immunoprecipitated with monoclonal anti-NPM1 antibody (1 μg per condition) and monoclonal anti-phospho STAT5 antibody (1:200) and probed with the indicated antibodies. (b) HEK 293T cells were plated in T-75 culture flasks on day 0 and transfected with either RFP-STAT5A or RFP-backbone vectors at the confluence of 80–90% on day 1, and the transfected cells were harvested and lysed on day 2. Lysate containing 2000 μg total protein was precipitated with 5 μl mouse monoclonal anti-phospho-STAT5 antibody (1:100) or 20 μl mouse IgG sepharose beads, mouse monoclonal anti-NPM1 antibody was used for detection. (c) The transfection of RFP-STAT5AY694F mutant vector followed the same procedure as the preceding panel. The lysate carrying 2000 μg total protein was precipitated with rabbit monoclonal anti-STAT5 antibody (1:50) or rabbit IgG XP Isotype control (1:50), mouse monoclonal anti-NPM1 antibody was used for detection

Fig 5: ACTH induces formation of Tregs through MC2R.(A) Comparison of MC1R, MC2R, MC4R, and MC5R gene expression by human CD4+CD25+ cells by qPCR (n = 3–5/group). (B) Comparison of MC2R gene expression by human CD3–CD19– cells, CD19+ B cells, CD3+ T cells, and CD4+CD25+ T cells by qPCR (n = 3–5/group). (C) Comparison of MC2R gene expression by human Tregs versus naive T cells by qPCR (n = 3/group). (D) Immunofluorescence staining of MC2R expression in mouse Tregs and non-Tregs from WT and CD28KO mice. Scale bar: 50 µm. (E) Flow cytometric analysis of percentage of CD4+CD25+Foxp3+ cells (Tregs) in Treg induction assay of splenocytes from WT and CD28KO mice, following either treatment with ACTH or no treatment (Control). (F) Comparison of gene expression of Il2ra and Il2 by Tregs of WT and CD28KO mice, following either ACTH treatment or no treatment (Control), by qPCR. (G) Comparison of MRAP2 protein expression by Tregs from CD28KO mice following ACTH treatment or no treatment (Control) by Western blot (P = 0.07). (H) Comparison of p-STAT5 gene expression by Tregs from CD28KO mice following ACTH treatment or no treatment (Control) by Western blot. (I) Representative flow cytometric plots of CD25 expression by CD4+ cells in Treg induction assay following treatment with ACTH and MC2R blocker. (J) Flow cytometric analysis of percentage of CD25+CD4+ cells following treatment with ACTH, ACTH, and MC2R blocker, or no treatment (Control) (n = 3/group). Data presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 by 2-way ANOVA with Turkey’s multiple comparisons (A, B, and J) and Student’s t test (C, E–H).